Chromatin-based DNA damage response (DDR) pathways protect cells from genome and epigenome instability, which are hallmarks of cancer and are thought to drive cancer progression. This proposal addresses questions of fundamental importance for epigenetic and genetic mechanisms involved in cancer. Histone H2A variants are critical components of chromatin but how they promote epigenome and genome stability are poorly understood. For this proposal, we aim to use genetic systems using our histone H2A variant knockout/knockdown engineered human cells with complimentary proteomic analyses of histone variants to answer fundamental questions for how histone H2A variants promote the DDR to maintain genome stability. We also aim to provide insights into how histone H2A variants regulate radiation responses, a therapy often used in cancer treatments. These studies provide innovative and new approaches to these questions that can provide transformative insights into chromatin-based DNA damage responses mediated by histone H2A variants. We will first analyze the DDR function of histone H2A variants in response to radiation, by using human cells deleted or depleted for histone H2A variants including H2AX, H2AZ, macroH2A and H2A.Bbd. Our preliminary results indicate that histone H2A variants play vital and unique roles in these pathways. These studies are poised to provide critical insights into how histone H2A variants regulate the DDR and response to IR. We will then exploit our preliminary analyses identifying candidate protein-interacting factors for each histone H2A variant to identify DDR effector proteins for individual histone H2A variants. Finally, we will test our working hypothesis for how macroH2A promotes DNA repair by HR through the interactions with a new DDR factor that we have identified. These studies will provide an unparalleled view of how histone H2A variants shape the DDR to protect the genome integrity across structurally and functionally diverse chromatin landscapes in human cells. DNA damaging agents are a major class of chemotherapeutic agents including radiotherapy and the epigenome represents exciting new leads for drug discovery. Thus, this work exploits a combination of genetic, biochemical and cellular approaches in human cells to identify key DDR pathways that promote genome and epigenome integrity, as well as responses to cancer-relevant therapies including radiation and PARP inhibitors that can translate to improved treatments for human cancers.